title: Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP).
abstract: Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.
authors.affiliation: Department of Medicine and Center for Molecular Genetics, University of California at San Diego, San Diego, California 92161-9111, USA.
authors.forename: L
authors.initials: L
authors.lastname: Taupenot
authors.forename: S K
authors.initials: SK
authors.lastname: Mahata
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: D T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism*; D019439:Chromaffin Cells / Q000378:metabolism*; D053379:Chromogranin A; D002864:Chromogranins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000378:metabolism* / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000378:metabolism* / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: C495403:Adcyap1 protein, rat; D002395:Catecholamines; D053379:Chromogranin A; D002864:Chromogranins; D017362:Cyclic AMP Response Element-Binding Protein; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D011493:Protein Kinase C; D002118:Calcium; D009638:Norepinephrine